Dataset Information

The LIM-homeodomain transcription factor LHX4 is required for the differentiation of retinal rod bipolar cells and rod-connecting cone bipolar cells

ABSTRACT: Study the role of the LIM-homeodomain transcription factor LHX4 in the development of retinal bipolar cell subtypes

ORGANISM(S): Mus musculus

PROVIDER: GSE126942 | GEO | 2019/02/23

REPOSITORIES: GEO

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Project description:Purpose: MÃ¼ller glia are the only glial cell type produced by the neuroepithelial progenitor cells which generate the vertebrate retina. MÃ¼ller glia are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, they function as an adult stem cell, mediating retinal regeneration among select vertebrate classes. However, the mechanisms which regulate MÃ¼ller development are poorly understood as considerable overlap exists in gene expression between retinal progenitor cells and differentiated MÃ¼ller glia. We investigate the functional role of the LIM homeodomain transcription factor Lhx2 in the specification and development of MÃ¼ller glia in the mouse. Methods: RNA-Seq was performed in collaboration with the Johns Hopkins School of Medicine Deep Sequencing and Microarray Core Facility. Libraries were prepared using Illumina TruSeq RNA Sample kit (Illumina, San Diego, CA) following manufacturerâs recommended procedure. The PCR amplified library was purified using RNAClean XP magnetic beads (Agencourt, Beverley, MA) and run out on a High Sensitivity DNA Chip (Agilent, Santa Clara, CA) for quality check. We used STAR to align RNA-Seq reads onto Ensembl mouse genome GRCm38, release 72. To generate the stand attribute for alignments containing splice junctions, we used the outSAMstrandField intronMotif program. The spliced alignments without strand definition were removed. Number of reads mapped to exons was counted by htseq-count. Genes expressed at very low levels were omitted from further analysis. Gene expression differences between wildtype and mutant samples, significance (p-value) and false discovery rate (FDR) were computed using the generalized linear models based EdgeR. Results: We observed a substantial reduction in expression of Notch pathway genes including Notch1, the Notch ligands Dll1 and Dll3, as well as gliogenic Notch effector genes such as Hes1, Hes5, Id1 and Sox8 and the MÃ¼ller-gliogenic factor Rax. We likewise observe a substantial reduction in expression of progenitor-specific genes such as Vsx2 and Fgf15. Furthermore, we observed a decrease in the expression of early-onset glial markers such as Crym , Spon1, and Car2. Retinal mRNA profiles of post-natal day 0.5 (P0.5) Lhx2 wild type (N=3) and Lhx2lox/lox; PdgfrÎ±-Cre ÎcKO (N=3) mice were generated using Illumina TruSeq and analyzed with Agilent high sensitivity DNA analsis kit.

Project description:Purpose: Müller glia are the only glial cell type produced by the neuroepithelial progenitor cells which generate the vertebrate retina. Müller glia are required to maintain retinal homeostasis and support the survival of retinal neurons. Furthermore, they function as an adult stem cell, mediating retinal regeneration among select vertebrate classes. However, the mechanisms which regulate Müller development are poorly understood as considerable overlap exists in gene expression between retinal progenitor cells and differentiated Müller glia. We investigate the functional role of the LIM homeodomain transcription factor Lhx2 in the specification and development of Müller glia in the mouse. Methods: RNA-Seq was performed in collaboration with the Johns Hopkins School of Medicine Deep Sequencing and Microarray Core Facility. Libraries were prepared using Illumina TruSeq RNA Sample kit (Illumina, San Diego, CA) following manufacturer’s recommended procedure. The PCR amplified library was purified using RNAClean XP magnetic beads (Agencourt, Beverley, MA) and run out on a High Sensitivity DNA Chip (Agilent, Santa Clara, CA) for quality check. We used STAR to align RNA-Seq reads onto Ensembl mouse genome GRCm38, release 72. To generate the stand attribute for alignments containing splice junctions, we used the outSAMstrandField intronMotif program. The spliced alignments without strand definition were removed. Number of reads mapped to exons was counted by htseq-count. Genes expressed at very low levels were omitted from further analysis. Gene expression differences between wildtype and mutant samples, significance (p-value) and false discovery rate (FDR) were computed using the generalized linear models based EdgeR. Results: We observed a substantial reduction in expression of Notch pathway genes including Notch1, the Notch ligands Dll1 and Dll3, as well as gliogenic Notch effector genes such as Hes1, Hes5, Id1 and Sox8 and the Müller-gliogenic factor Rax. We likewise observe a substantial reduction in expression of progenitor-specific genes such as Vsx2 and Fgf15. Furthermore, we observed a decrease in the expression of early-onset glial markers such as Crym , Spon1, and Car2.

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The LIM-homeodomain transcription factor LHX4 is required for the differentiation of retinal rod bipolar cells and rod-connecting cone bipolar cells

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